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Live imaging of a transgenic Tribolium castaneum (red flour beetle) embryo expressing mEmerald-labeled H2A-H2B nanobodies under control of the endogenous tubulin 1-alpha promoter.

The transgenic Tribolium line was created by integrating the H2A-H2B nanobody sequence into the AGameOfClones vector concept (Strobl et al. 2018, eLife), respective embryos were imaged using light sheet-based fluorescence microscopy (Strobl et al. 2015, Nature Protocols).(A) A Tribolium embryo during germband elongation stage. The maximum projection along the optical axis of the detection objective (ZP), as well as five single planes (PL) at different depths show that mEmerald-labeled nanobodies translocate to the nuclei. The metaphase plate as well as anaphasic sister chromatids are accurately identifiable in different cells during mitosis (detail image).(B) Long-term live imaging of a Tribolium embryo from germband elongation until the onset of muscular movement. The mEmerald-labeled H2A-H2B nanobodies provide a high signal-to-noise ratio even after the acquisition of more than 800 image stacks, i.e. 120,000 single planes, over a period of five days. Upon completion of observation, the embryo was removed from the microscope, placed in growth medium and developed into a morphologically intact adult beetle. Illumination: 2.5× NA 0.06 EC Epiplan-Neofluar, 135 µW laser power, exposure time of 50 ms. Detection: objective 10× NA 0.3 W N-Achroplan, 525/50 bandpass filter, Andor Clara CCD camera. The data were neither de-convolved nor fused along multiple directions.